PROJECT SUMMARY/ABSTRACT Herpes simplex virus (HSV) type 1 is a leading cause of infectious corneal blindness. It causes eye disease by reactivation from a latent viral reservoir in corneal nerves. Despite extensive research, the mechanisms that regulate HSV infection in neurons are not well characterized. A better understanding of this is critical for identifying new therapeutic strategies. We have developed a novel culture system, using dissociated adult murine trigeminal ganglia (TG), for studying HSV infection in neurons. Preliminary data from our lab indicates that, unlike its role in replicating cells, the viral immediate early (IE) gene product, ICP27, restricts productive viral infection in neurons, especially in A5+ ganglionic neurons, and promotes viral latency. In the current proposal we will further characterize this novel function for ICP27, as well as study the mechanisms by which ICP27 accomplishes these functions. In the first two specific aims of this proposal, we will characterize the role that ICP27 plays in restricting productive infection and promoting viral latency, using ICP27 null mutants, an ICP27 promoter mutant with delayed kinetics of expression, and viral mutants with deletions in different ICP27 functional domains. We will further characterize the role of ICP27 in restricting infection in neurons through the use of novel AAV vectors for the efficient transduction of sensory neurons with ICP27. Our preliminary data also suggests that VP16, a late viral protein in replicating cells, is expressed very early in TG neurons, and that ICP27 restricts transcription of both VP16 and ICP4 in cultured TG neurons. In the third specific aim we will further characterize ICP27 mediated inhibition of ICP4 and VP16 transcription and test hypotheses about the way in which this is achieved. Finally, we will test hypotheses that ICP27 restricts productive infection in A5+ neurons, in part, by restricting ICP4, VP16 and HCF1 to the cytoplasm, thus preventing transactivation of viral IE genes. These concepts and studies are innovative, and are a result of being able to directly study HSV infection in neurons, as well as being able to differentiate the outcome of infection in A5+ neurons, the major site of HSV latency. The outcome of these studies should generate new insights into the mechanisms regulating HSV infection of neurons; the first step in developing new therapy strategies.